The proposed research consists of two sections, the chemistry of the amatoxins with respect to their specific complexation with eukaryotic RNA polymerases and the application of selected amatoxin derivatives to study the transcriptive functions of eukaryotic RNA polymerase. Examination of the amatoxin chemistry will focus initially upon electrophilic substitution in the 6-hydroxy-2-sulfoxy indole moiety of alpha-amanitin. The purified derivatives are to be tested for their inhibition properties on eukaryotic RNA polymerases I, II, and III. Those amatoxin derivatives with Ki less than 10 to the minus 6th power M for RNA polymerase III or I are to be radiolabelled, preferably with 125I, and characterized with respect to KD and half-life of the amatoxin-RNA polymerase complex. Structural assignments for each derivative are to be obtained by comparison with previously defined compounds and by proton magnetic resonance. The first objective of this research is to obtain high specific radioactivity derivatives suitable for quantitation of eukaryotic RNA polymerase III and I by a ligand binding assay. The second objective of this research is to obtain derivatives useable in affinity isolation of RNA polymerases II and III. These derivatives of alpha-amanitin will provide the vehicle to probe transcriptive functions in the RNA polymerase; the (125I)-7-iodo-alpha-amanitin provides a ligand binding assay for the quantitation of RNA polymerase II. The association and dissociation rates for RNA polymerase II binding to DNA will be determined by this assay in order to estimate the KD for the RNA polymerase-DNA binary complex. The effect of the DNA structure (e.g., double-stranded, single-stranded, haplotomic scissions) on the kinetic parameters will be established. Using an affinity isolation derivative of alpha-amanitin, the RNA polymerase II from nuclei, along with neighboring chromosomal proteins, will be purified. The chromosomal proteins will be tested for promotion of limited and high affinity binding of RNA polymerase II to DNA.